Macromolecules such as LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds are useful for the prevention and treatment of infections and infestations caused by helminths, nematodes, acarids and endo- and ectoparasitic arthropods when parenterally administered to warm-blooded animals. Parenteral compositions are sterilized prior to administration to an animal. Gamma radiation is an effective sterilization process for eliminating microbial contaminants. However, certain macromolecules such as LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds degrade and lose much of their biological activity when irradiated. This destructive and degradative response to gamma radiation precludes the use of gamma radiation as a means to sterilize certain macromolecule-containing compositions.
It is an object of the present invention to provide stable compositions which can be irradiated that are useful for the prevention or treatment of infections and infestations caused by helminths, nematodes, acarids and endo- and ectoparasitic arthropods when parenterally administered to warm-blooded animals.
It is also an object of this invention to provide a method for introducing and maintaining levels of LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds in the blood of warm-blooded animals for extended periods of time.
The present invention relates to stable microsphere compositions which can be irradiation sterilized for parenteral administration. The compositions comprise, on a weight basis, about 20% to 95% of a fat, a wax or a mixture thereof, about 1% to 50% of an LL-F28249xcex1-xcex compound, a 23-oxo or 23-imino derivative of an LL-F28249xcex1-xcex compound, a milbemycin compound or an avermectin compound, 0 to about 30% of an oil, semi-soft fat, fatty acid derivative or mixtures thereof and about 0.001% to 10% of an antioxidant.
Surprisingly, it has been found that the microsphere compositions of the invention can be sterilized by gamma radiation without degradation of its biological activity. Also unexpectedly, the microsphere compositions can achieve an effective sustained release effect of the water-insoluble, complex macrolides.
In accordance with the present invention, the stable, slow release microsphere compositions comprise an LL-F28249xcex1-xcex compound, a 23-oxo or 23-imino derivative of an LL-F28249xcex1-xcex compound, a milbemycin compound or an avermectin compound; a fat, a wax or a mixture thereof; an antioxidant and, optionally, an oil, a semi-soft fat, a fatty acid derivative or a mixture thereof. The microsphere compositions are parenterally administered by dispersion in a pharmaceutically and pharmacologically acceptable liquid vehicle. The invention also provides a method for introducing and maintaining blood levels of an LL-F28249xcex1-xcex compound, a 23-oxo or 23-imino derivative of an LL-F28249xcex1-xcex compound, a milbemycin compound or an avermectin compound in warm-blooded animals for an extended period of time.
Preferred stable microsphere compositions of the invention comprise, on a weight basis, about 50% to 90% of a fat, a wax or a mixture thereof; about 5% to 25% of an LL-F28249xcex1-xcex compound, a 23-oxo or 23-imino derivative of an LL-F28249xcex1-xcex compound, a milbemycin compound or an avermectin compound; 0 to about 20% of an oil, a semi-soft fat, a fatty acid derivative or mixtures thereof and about 0.01% to 5% of an antioxidant.
The compounds designated LL-F28249xcex1-xcex are (collectively) isolates from the fermentation broth of the microorganism Streptomyces cyaneogriseus subspecies noncyanogenus, deposited in the NRRL under deposit accession No. 15773. The method for preparation of LL-F28249xcex1 is disclosed in U.S. Pat. No. 5,106,994 and its continuation, U.S. Pat. No. 5,169,956, which are incorporated herein by reference thereto.
The LL-F28249xcex1-xcex compounds are represented by the following structural formula: 
The 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, useful in the stable microsphere compositions of this invention, are disclosed in U.S. Pat. No. 4,916,154 which is incorporated herein by reference thereto.
A preferred LL-F28249xcex1-xcex compound and 23-imino derivative of an LL-F28249xcex1-xcex compound useful in the microsphere compositions of this invention have the following structural formulas: 
and 
The wax of the present invention may be defined as set forth in Hawley""s The Condensed Chemical Dictionary, Eleventh Edition, as a low-melting organic mixture or compound of high molecular weight, solid at room temperature and generally similar in composition to fats and oils except that it contains no glycerides. Some are hydrocarbons; others are esters of fatty acids and alcohols. These compounds include saturated or unsaturated long chain C10-C24 fatty acids, alcohols, esters, salts, ethers or mixtures thereof. They are classed among the lipids. Waxes are thermoplastic, but since they are not high polymers, they are not considered in the family of plastics. Common properties are water repellency; smooth texture; nontoxicity; freedom from objectionable odor and color. They are combustible and have good dielectric properties. They are soluble in most organic solvents and are insoluble in water. The major types are as follows:
I. Natural
1. Animal (beeswax, lanolin, shellac wax, Chinese insect wax)
2. Vegetable (carnauba, candelilla, bayberry, sugar cane)
3. Mineral
(a) Fossil or earth waxes (ozocerite, ceresin, montan)
(b) petroleum waxes (paraffin, microcrystalline) (slack or scale wax)
II. Synthetic
1. Ethylenic polymers and polyol ether-esters (Carbowax(copyright), registered trademark of Union Carbide Corp., New York, N.Y.)
2. Chlorinated naphthalenes
3. Hydrocarbon type via Fischer-Tropsch synthesis
The fat of the invention may be defined as set forth in Hawley""s The Condensed Chemical Dictionary, Eleventh Edition, as a glyceryl ester of higher fatty acids such as stearic and palmitic. Such esters and their mixtures are solids at room temperatures and exhibit crystalline structure. Lard and tallow are examples. The term xe2x80x9cfatxe2x80x9d usually refers to triglycerides specifically, whereas xe2x80x9clipidxe2x80x9d is all-inclusive.
The fat is preferably a hard fat composed of mono-, di- or triglyceryl esters of long chain C10-C24 fatty acids. The mono-, di- or triglycerides are composed predominantly of stearates, palmitates, laurates, linoleates, linolenates, oleates, and residues or mixtures thereof; those having melting points greater than 50xc2x0 C. are most preferred (i.e. a xe2x80x9chigher meltingxe2x80x9d fat). Glyceryl tristearate is a most preferred fat. Additionally, lipophilic salts of fatty acids such as magnesium stearate and the like are also suitable.
The oil, semi-soft fat or fatty acid derivative of the invention are agents which are soluble in the molten hard fat and which accelerate physical transformation of the hard fat crystal from less stable forms to more stable forms at or near room temperature after the molten hard fat or wax solidifies. Preferably, the oil or semi-soft fat may include mixtures or relatively pure forms of mono-, di- or triglyceryl esters with short to medium fatty acid chain lengths, that is, C2 to C18. Semi-soft fats refer to glyceryl esters having melting points at or near room temperature. Fatty acid derivatives include short and medium chain length fatty acids, alcohols, esters, ethers, salts or mixtures thereof; those having melting points less than 50xc2x0 C. are most preferred (i.e., a xe2x80x9clower meltingxe2x80x9d fat). Glyceride oils and semi-soft fats are particularly suitable because they are physiological constituents of the body and are biocompatible and biodegradable.
The antioxidant of the invention may be defined as set forth in Hawley""s The Condensed Chemical Dictionary, Eleventh Edition, as an organic compound added to rubber, natural fats and oils, food products, gasoline and lubricating oils to retard oxidation, deterioration, rancidity and gum formation, respectively. Rubber antioxidants are commonly of an aromatic amine type, such as di-xcex2-naphthyl-p-phenylenediamine and phenyl-xcex2-naphthylamine. Many antioxidants are substituted phenolic compounds (butylated hydroxyanisole, di-tertbutyl-p-cresol and propyl gallate). Food antioxidants are effective in very low concentration and not only retard rancidity but protect the nutritional value by minimizing the breakdown of vitamins and essential fatty acids.
Surprisingly, the antioxidant of the invention allows the present microsphere compositions to be sterilized with gamma radiation and maintain shelf life without significant loss of biological activity. In the absence of an antioxidant, macromolecules such as LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds degrade and lose much of their biological activity when irradiated. Antioxidants suitable for use in the microsphere compositions of the invention include, but are not limited to, butylated hydroxytoluene, butylated hydroxyanisole, tert-butylhydroxyquinone, propyl gallate, a-tocopherol, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and the like, with butylated hydroxytoluene being a preferred antioxidant.
The stable microspheres of the invention are dispersed in a pharmaceutically and pharmacologically acceptable liquid to obtain a slow release composition for parenteral administration. The vehicle may be aqueous buffered systems or oil systems. The oil system may be derived from animal or vegetable sources or be synthetic. Preferred vehicles include neutral mono-, di-or triglyceride liquid or mixtures thereof. A neutral oil is one containing no residual acid. Vehicles suitable for use in the compositions of this invention include aqueous systems such as buffered salines; organic solvents such as glycols and alcohols; and water immiscible liquids such as oils, depending upon the solubility of the active ingredient being administered.
Excipients such as thickening agents, surfactants, salts, buffers or mixtures thereof may be included in the compositions of the invention. The amounts of said excipients suitable for use in the invention range from about 0.1% to 20% on a weight basis.
Blood levels of the LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds may be obtained and maintained for extended periods of time by injecting animals with the compositions of the invention in a suitable vehicle. Unexpectedly, the water-insoluble macrolide antibiotics can be effectively delivered from the microsphere formulation for a sustained release effect.
Maintained blood levels of the active compounds are associated with the protection or treatment of warm-blooded animals against infections and infestation by helminths, nematodes, acarids and endo- and ectoparasitic arthropods. Maintaining the blood levels is an indication of the slow release of the active ingredient. The invention includes the use of the compositions herein to introduce and maintain levels of LL-F28249xcex1-xcex compounds, 23-oxo and 23-imino derivatives of LL-F28249xcex1-xcex compounds, milbemycin compounds and avermectin compounds in the blood stream of animals.
When parenterally administered, the compositions of this invention are highly effective for protecting or treating warm-blooded animals such as dogs, cats, cattle, sheep, horses, swine, goats, poultry and the like against infection and infestation by helminths, nematodes, acarids and endo- and ectoparasitic arthropods.
Helminthiasis is a widespread disease found in many animals and is responsible for significant economic losses throughout the world. Among the helminths most frequently encountered are the group of worms referred to as nematodes. The nematodes are found in the intestinal tract, heart, lungs, blood vessels and other body tissues of animals and are a primary cause of anemia, weight loss and malnutrition in the infected animals. They do serious damage to the walls and tissue of the organs in which they reside and, if left untreated, may result in death to the infected animals.
The nematodes most commonly found to be the infecting agents of ruminants include Haemonchus and Ostertagia generally found in the abomasum; Cooperia, Trichostrongylus and Nematodirus generally found in the intestinal tract, and Dictyocaulus found in the lungs. In non-ruminant animals important nematodes include Toxocara and Ancylostoma in the intestine and Dirofilaria in the heart of dogs; Ascaris in the intestine of swine; Ascaridia and Heterakis in the intestine of poultry; and large and small strongyles in equines. Treatment of animals to prevent infestation thereof by the above nematodes or to reduce or control the proliferation of these infecting agents in animals is thus an important and desirable advantage of the present invention.
Besides controlling helminths and nematodes, the present invention also controls arthropod endo-parasitic infestations such as cattle grub and ecto-parasitic infestations such as psoroptic mange and cattle ticks.
The microspheres of the invention may be prepared by incorporating the active ingredient, antioxidant and other excipients with a molten fat, wax or mixture thereof, optionally admixing the oil, semi-soft fat, fatty acid derivative or mixtures thereof, and then forming microspheres of the resulting mixture by a variety of techniques such as emulsifying or atomizing the mixture or by processing the mixture of ingredients and molten fat, wax or mixture thereof mechanically and cooling, for example, utilizing a centrifugal disc.
Alternatively, the mixture of active ingredients, antioxidants, excipients and fat, waxes and mixtures thereof and oil, may be cooled to give a solid which may then be processed by procedures such as milling, grinding and the like. The microspheres, preferably fat microspheres, may be up to 1,000 microns in diameter, with a weight average size range of about 25 microns to 300 microns being preferred for parenteral administration.